The objective of this project is to understand how enzymes utilize conformational fluctuations to facilitate catalysis. Over the past 3 decades it has become increasingly clear that rather existing as static structures, proteins are actually ensembles of sometimes very different conformational states, and the fluctuations are critical to function. It is of great import to know how this is done. Are there unifying principles that connec proteins with different functions? Here we take advantage of two discoveries by our group during previous funding cycles, which demonstrates that the enzyme adenylate kinase (AK) from E. coli, uses local unfolding to modulate its enzymatic activity - in effect, the energy landscape has unfolding within its functionally important repertoire. Initially we found unfolding to occur only in the LID domain and that this unfolding modulated Km at physiological conditions. More recently we found unfolding in other parts of the molecule, demonstrating that local unfolding in some regions modulates Km, local unfolding in other regions modulates kcat, and that unfolding mediates communication between domains. This mode of conformational change stands in stark contrast to the current accepted model, whereby a rigid-body hinge opening and closing reaction is believed to facilitate catalytic turnover. In the proposal, we leverage this unfolding reaction into a mutation strategy designed to investigate the coupling between the different regions of AK, and how that coupling produces an ensemble of states of AK that changes during the course of catalytic turnover. Our approach builds on our previous results and is geared toward the development of a quantitative experimentally derived model of AK. We will perform binding and stability measurements using isothermal titration calorimetry (ITC), circular dichroism (CD) monitored thermal unfolding and hydrogen exchange (HX), and we will monitor the kinetics of the conformational and enzymatic processes using NMR 15N relaxation dispersion (CPMG) and steady state enzymatic analysis.